The objective of the proposed research is to enhance the understanding of mechanisms which regulate the alternative splicing process. This differential post-transcriptional processing is important in the regulated expression of structurally distinct protein isoforms, essential to eukaryotic development and cell differentiation. The detailed mechanisms which dictate how alternative splicing is accomplished, however, remain obscure. As such, model systems are needed in which the factors contributing to these mechanisms can be dissected. Tropomyosin, a ubiquitous protein, plays an important role in the regulation of contractility of muscle and nonmuscle cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and non-sarcomeric muscle, it is a component of the thin filament. The alpha-tropomyosin (alpha-TM) gene in the rat exhibits an unprecedented capacity to generate isoform diversity through differential exon splicing in a tissue-specific and developmentally-regulated manner. These isoforms, produced by alternative splicing, are specifically expressed in nonmuscle, smooth and striated (cardiac and skeletal) muscle cells. This system offers the opportunity to study the mechanisms and factors which are of prime importance in determining developmental and tissue specificity in regulating alternative RNA splicing. The specific aims of this project are (1) expanding the identification of the alpha-TM mRNA products; (2) characterization of the alpha-TM alternative splicing mechanisms; and (3) continuing the characterization of nerve extract factors which can regulate alpha-TM post-transcriptional processing. The second aim will be accomplished by constructing a set of plasmids incorporating either the complete genomic sequence or subsegments of it in hybrid minigenes. Expression of these constructs will be obtained by transformation of nonmuscle, as well as differentiated and undifferentiated muscle cells in culture, followed by analysis of their products. The third aim will focus on defining the biochemical properties of exogenous neural factors which can regulate alpha-TM isoform production. Future investigations will focus on the mechanisms by which these neural-specific factors illicit their effects by combining extracts with alpha-TM minigene constructs in cellular transformation studies.